First 4 Plates_RFS_Run1

Normalize templates

Use plate reader to quantify templates

Normalize to between 20 ng/ul and 150 ng/ul

2017 alfalfa: Transfer to conical pcr plates. Normalize on nimbus to 100 ng/ul in 30 ul.

Templates:

Restriction Digestion

(Keep MM and reaction plates on ice)

Add 3 ul Digestion MM to 4 plates using the 8 channel

Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C for 45 minutes and 4C until stopped.

Adaptor Ligation

Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.

Add 1 ul of EcoR1 Adaptors with 8-channel or BenchSmart and Track which template plate gets which MID plate.

Label reaction plates with MID plate used.

Cover, vortex, and spin plates. Incubate at 16C for 6 hours on Thermocycler.

Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.

PCR Amplification

Create two working pools. For pool 1A, pool ligated plates matching well to well in sets of 4. For pool 1B, pool ligated plates with plate 2 and plate 4 flipped upside down (H12 in A1). (Mix together 20 ul from each member of pool in new plate and then use 4 ul from that plate for pcr template). We are doing duplicate pcr.

GBS1-4_PCR_A: Pool with exact replicate well to well

GBS1-4_PCR_B: Pool with flipped plates

 Add 16 ul of PCR1 MM to each well of plates

Add 4 ul of restriction/ligation products to each well

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

*pause step

Extra PCR

Add 2.125 ul of the MM directly to the previous PCR

Then continue with the last four unlisted steps for GBS1 from above:

Comparisons

qPCR GBS Plates 1-4 Pool PCR A and Pool PCR B

• Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

• Combine 5uL from each well of GBS1-4_PCRA into a pool. Make a 1:1000 dilution of this pool to run on qPCR.

• Combine 5uL from each well of GBS1-4_PCRB into a pool. Make a 1:1000 dilution of this pool to run on qPCR.

• After 1:1000 pools have been made for each PCR plate, combine PCRA and PCRB undiluted pools together into one tube and label GBS1-4. Make a 1:1000 dilution of this pool to run on qPCR.

• Add 16 ul of Illumina Library Quantification MasterMix to each well:

• Add 4 ul of template, pool, or standards to each well:

Results:

Average quantities observed for all products:

GBS1-4 PCR_A Col_1: will be repeated on next qPCR

GBS1-4 PCR_A Col_4: 74.74 nanomoles

GBS1-4 PCR_A Col_10: 78.98 nanomoles

GBS1-4 PCR_B Col_1: will be repeated on next qPCR
GBS1-4 PCR_B Col_4: 66.37 nanomoles

GBS1-4 PCR_B Col_10: 44.6 nanomoles

GBS1-4 PCR_A Pool: 106.5 nanomoles

GBS1-4 PCR_B Pool: 90.94 nanomoles

GBS1-4 Pool: 109.05 nanomoles

The full results report can be found below:

Open

Bioanalyzer

BioAnalyzer Chip Set up:

Well 1: GBS1-4 PCR_A Pool

Well 2: GBS1-4 PCR_B Pool

Well 3: GBS1-4 Pool

Well4: GBS1-4 PCR_A F3

Well 5: GBS1-4 PCR_A B5

Well 6: GBS1-4 PCR_A G7

Well 7: GBS1-4 PCR_A H12

Well 8: GBS1-4 PCR_B C1

Well 9: GBS1-4 PCR_B A4

Well 10: GBS1-4 PCR_B D6

Well 11: GBS1-4 PCR_B H8

Well 12: GBS1-4 PCR_B E11

Electropherogram of all wells:

Open

Electropherogram comparing wells 1-4:

Open

Electropherogram comparing wells 5-8:

Electropherogram comparing wells 9-12:

Gel:

iSeq